WO2004016806A1 - Utilisation d'une polymerase dotee d'une activite de 3'-5' exonuclease pour effectuer une analyse de sequence de genes - Google Patents

Utilisation d'une polymerase dotee d'une activite de 3'-5' exonuclease pour effectuer une analyse de sequence de genes Download PDF

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WO2004016806A1
WO2004016806A1 PCT/CN2003/000695 CN0300695W WO2004016806A1 WO 2004016806 A1 WO2004016806 A1 WO 2004016806A1 CN 0300695 W CN0300695 W CN 0300695W WO 2004016806 A1 WO2004016806 A1 WO 2004016806A1
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Prior art keywords
primer
polymerase
exonuclease activity
primers
sequence analysis
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PCT/CN2003/000695
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English (en)
Chinese (zh)
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Kai Li
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Zhang, Xu
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Priority to JP2004528265A priority Critical patent/JP2005535336A/ja
Priority to EP03787581A priority patent/EP1536019A4/fr
Priority to AU2003257798A priority patent/AU2003257798A1/en
Publication of WO2004016806A1 publication Critical patent/WO2004016806A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing

Definitions

  • the present invention relates to a method for gene sequencing, and specifically refers to the use of a polymerase having 3 'to 5' exonuclease activity in combination with a specific 3 'terminal modified primer for rapid sequencing of a gene's base sequence.
  • the current reliable genetic assay method uses a polymerase without 3 'to 5' exonuclease activity for primer extension reaction.
  • the gene analysis process is complicated, and each signal can only provide up to one base of information, which is more prominent.
  • the problem is that the existing methods are difficult to perform high-throughput analysis of single base polymorphisms.
  • existing methods can achieve all-gene sequencing, more economical and faster gene sequencing technology has become a hotspot for competition in various countries. For example, Airfymetrix's oligonucleotide hybridization chip has some research value.
  • Another method of sequencing is the end-stop method, which is the main method of modern gene sequencing. Although this method is very reliable, because each signal can only provide information about one base at most, the sequencing speed is limited. Importantly, this method requires a known sequence of genes as the basis for sequencing. This dependence on known sequences greatly slows down the speed of this method in determining unknown sequences. In addition, the amplification efficiency of this method is limited, and it is often necessary to amplify the gene to be tested first. Obviously, this method has the disadvantage of being time-consuming, but it is actually an indirect sequencing method.
  • Oligonucleotide hybridization sequencing chips can also be used for sequencing, but due to the inherent thermodynamic heterogeneity of hybridization technology, its accuracy is not high. It is still in the laboratory research and improvement stage, and its practicality remains to be seen.
  • all current enzymatic methods related to sequencing include the classic terminal termination method and the recently developed single base extension method. Their labeling pathways are through dideoxy triphosphate nucleotides, because dideoxy triphosphate nucleotides cannot be dehydrated. It has the effect of stopping extension, and the reaction is a one-way primer extension process.
  • the use of dideoxytriphosphate in a unidirectional primer extension reaction causes the amplification efficiency of primer extension to be only proportional to the number of reaction cycles. In a two-way primer extension reaction without dideoxytriphosphate, the efficiency is proportional to the square of the number of reaction cycles. Summary of the invention
  • the purpose of the present invention is to overcome the deficiencies in the prior art, and to provide an application
  • a 3 'to 5' exonuclease-active polymerase performs gene sequence analysis, and the present invention can detect single base polymorphisms and unknown gene sequences.
  • a method for performing gene sequence analysis using a polymerase having 3 'to 5' exonuclease activity includes the following steps:
  • a polymer having 3 'to 5' exonuclease activity and a modified primer constitute a molecule "on / off", and a selective primer extension reaction is performed with the participation of said molecule "on / off";
  • the primer set is composed of a sub-set of primers with different 3 'terminal sequences.
  • the primer set is composed of an unmodified oligonucleotide and a modified oligonucleotide.
  • the product is a product without a labeled signal or a labeled signal.
  • the labeled signal of the product comes from a labeled primer or from a labeled nucleotide triphosphate substrate.
  • the modification refers to chemical modification of the oligonucleotide so that it has a labeled signal or changes the response characteristics of the primer to the nuclease.
  • Said molecule "on / off” can change the characteristic of 3 'terminal modification which is not resistant to exonuclease Determine the marker signal carried by the oligonucleotide primer set, or turn off the extension reaction of the specific oligonucleotide primer set that is resistant to the 3 'end modification of the exonase.
  • This method uses a polymerase with 3 'to 5' exonuclease activity and "on / off" consisting of modified primers for primer extension reaction.
  • the molecular switch is more accurate than conventional polymerases without correction function The primer extension reaction performed;
  • This method can be used for direct sequencing of unknown sequences
  • This method does not rely on a known sequence when sequencing
  • Each signal obtained by this method contains information of one or more base sequences.
  • This method provides a high-throughput analysis method for single base polymorphism analysis and gene sequence analysis.
  • FIG. 1 is a schematic diagram of the basic principle of the present invention
  • Example 3 is a gel electrophoresis diagram of Example 1 of the present invention.
  • Example 4 is a sequencing diagram of Example 1 of the present invention.
  • FIG. 5 is a measurement diagram of liquid scintillation counting in Example 2 of the present invention.
  • Example 6 is a gel electrophoresis diagram of Example 3 of the present invention.
  • Example 7 is a gel electrophoresis diagram of Example 4 of the present invention.
  • Example 8 is a gel electrophoresis diagram of Example 5 of the present invention.
  • FIG. 9 is a gel electrophoresis diagram of Example 5 of the present invention.
  • Fig. 10 is a schematic diagram of the molecules 'on / off' of the present invention.
  • 1 represents a polymerase-mediated primer extension with 3 'to 5' exonuclease activity.
  • Extension reaction 2 for ordinary primers, 4 for 3 'specific modified primers, 6 for products with no labeled signal or no products; 1 in FIG.
  • P represents the paired primers, and 1, 2, 3, 4, 5, 6 respectively represent primers -1, -2, -3 , -4, -5, -6 base unpaired primers;
  • PM indicates a paired primer
  • MM indicates an unpaired primer
  • 1 indicates a polymerization center
  • 2 indicates an enzymatic hydrolysis center
  • 3 indicates Primers
  • 4 means correction for unpaired primers
  • 5 means on effect
  • 6 means off effect
  • Y means meeting matching requirements
  • N means not matching Matching requirements.
  • a molecule "on / off" composed of a polymerase having 3 'to 5' exonuclease activity and a 3 'terminal modified primer is used to perform a primer extension reaction, and the difference between the pairing of the primer and the template or the incomplete pairing is used.
  • This method utilizes the highly specificity of the enzyme's proofreading effect during primer extension to directly obtain one or more base sequence information of the 3 'end or near 3' end of a known primer, thereby achieving a primer extension reaction. Rapid determination of polymorphic base sites of known sequences or unknown base sequences in templates.
  • the probability of a mutation occurring is about 2/1000.
  • a primer extension reaction of a polymerase with 'to 5' exonuclease activity can reduce the chance of mutations by 80-90%, thereby greatly increasing the number of correct products and reducing the number of incorrect products. This huge difference is known throughout the world in the process of DNA replication in animals, plants and bacteria.
  • the bigger, yet unknown, difference is in the first primer extension reaction.
  • the first step in primer extension determines whether the reaction changes the sequence of the primer.
  • a primer extension reaction is performed by a polymerase without a 3 'to 5' exonuclease activity, it will proceed in a manner that does not change the sequence of the primer, regardless of whether the primer and the template are completely paired. In this way, the pairing of the primer and the template may be distorted, that is, the sequence of the template cannot be accurately reflected from the primer sequence.
  • the primer extension reaction performed by a polymerase having 3 'to 5' exonuclease activity depends on the pairing, and if it is paired, the primer sequence is not changed by 100%; if it is not paired, the primer sequence is changed so that It matches the template exactly. It can be seen that if a polymerase with 3 'to 5' exonuclease activity can reasonably be used to distinguish pairing conditions during primer extension reactions, the template sequence can be read accurately from the primer sequence.
  • the modification of the primer includes: (1) labeling the 3 'terminal nucleotide of the primer, and the product of the primer that has been completely paired retains the labeled nucleotide, and the product of the primer that is not completely paired is the labeled nucleoside.
  • the acid is cleaved by the 3 'to 5' exonuclease correction function without a labeled signal; (2) the use of a modified 3 'terminal nucleoside Acid, to cause insensitivity to 3 'to 5' exonuclease, to achieve a perfectly matched primer has an extension product and a 3 'or near 3' terminal incompletely matched primer has no extension effect. (see picture 1 )
  • the extension of the paired primers has nothing to do with the fidelity of the polymerase used, or whether the 3 'end of the paired primers can be degraded by exonase.
  • the extension and otherwise depend on the fidelity of the polymerase used and the extent to which the 3' end of the unpaired primer is degraded by the exonase.
  • Efficacy of "on” and “off” in a composite molecular switch For paired primers, the polymerization reaction is directly performed in the polymerization center of the enzyme, that is, the "on” effect, as shown in Fig. 10; Primers are transferred from the polymerization center of the enzyme to the 3 'to 5' exonuclease digestion center. Due to the characteristics of the 3 'end exonuclease modified by the primer, a long-term non-enzymatic product appears. During the enzymatic hydrolysis process, the DNA polymerization reaction was "closed” due to the idling of the polymerization center, as shown in Figure 10. This paired primer is extended, and the unbindable primer does not extend the binary effect of presence or absence, which perfectly satisfies the binary identification of specific sites in the single-base polymorphism analysis.
  • the products are expressed as polymerases with detectable signals and without detectable signals, that is, 3 'to 5' exonuclease activities Turning off the detectable signal of the unpaired primer is actually an 'on / off' effect on the presence or absence of the detectable signal.
  • the signal source of the present invention differs depending on the labeling method. There are three main labeling methods that can be used, namely the 3 'terminal base of a primer in a primer set, a labeled nucleotide triphosphate substrate, and a labeled paired primer.
  • a primer that is fully paired with the template will produce a product containing the labeled signal.
  • a label may be a radioactive label or a non-radioactive label such as a fluorescent label.
  • a primer that is incompletely paired with the template will have two possibilities depending on the modification of the primer, that is, no product is generated due to base mismatches, or no polymerase is generated due to the correction function of the polymerase from 3 'to 5' exonuclease Marked products.
  • the ratio of the labeled triphosphate nucleotide substrate to the unlabeled tribasic acid nucleotide substrate varies depending on the type of the label, and the variation varies between Between 1: 10 and 1: 100.
  • the label can be at its 5 'end, or at any other base than the 3' end.
  • Primer design for detection of single base polymorphisms Generally, two forward primers are designed at one site, and one reverse primer is a primer set. The specific number depends on the actual needs.
  • the directional primers are fully matched and incompletely matched with the template, that is, a sub-set of primers.
  • the labeling method is selected according to the experimental conditions and detection methods, and designed and synthesized by the biosynthesis company. For example, the following primers are designed:
  • X represents a base paired with a template
  • Y represents a base not paired with a template
  • X, Y are unmodified bases
  • X, 2 are modified bases.
  • Primer design for detection of unknown gene fragments A mathematical model for sequence analysis using a DNA polymerase with 3 'to 5' exonuclease activity to recognize base sequences.
  • the primer set depends on the determined number of bases at the 3 ′ end, and is composed of four bases with a 5 ′ end of 4: a four-base 1: 1 1: 1: 1 swing sequence and 3 ′ a sequence-specific primer.
  • the following 3 'end of two bases determined set of primers comprising 42 primers, i.e., primer 16 (X represents the wobble base).
  • a polymer having "3 'to 5' exonuclease activity and a modified primer constitute a molecule" on / off ", and a selective primer extension reaction is performed with the participation of said molecule" on / off ";
  • the specific separation method depends on the specific application method. When the present invention is applied to electrophoresis, the separation process can be omitted; when the present invention is applied to a biochip, the washing under stringent conditions will remove the labeled signal on non-DNA molecules. If the labeling signal originates from labeled primers and the background noise is too high, you can incubate with a low concentration of exonuclease for 15 minutes to 1 hour at 25 to 37 degrees Celsius. The labeled signal on the DNA molecule can be visualized and detected by corresponding conventional methods, and the specific method is determined by the labeling method. For enzyme labels or other chemical reactant labels such as digoxigenin and biotin, a specific chemical imaging reaction is required before detecting the signal.
  • the labeling method is a method that cannot be directly scanned and detected, such as sulfurization, ethidium bromide or GYBR staining can be used to analyze the results under gel electrophoresis, or the results can be analyzed by mass spectrometry. Make judgments. If the labeling method is radioactive or direct fluorescence method, no imaging process is required, and the radioactive intensity or fluorescent intensity can be directly scanned for measurement or the auto-imaging method is used for subsequent scanning analysis of the image. If the labeling method is chemiluminescence or indirect fluorescence, you need to perform pre-blocking of development, imaging reaction, and washing process after the house image, and then detect the labeling signal.
  • the labeling method is chemiluminescence or indirect fluorescence, you need to perform pre-blocking of development, imaging reaction, and washing process after the house image, and then detect the labeling signal.
  • the detected label signal directly represents the specific fluorenyl sequence at the 3 'end of the primer at the corresponding site. Sequence analysis can be performed manually or computer-assisted. Whether a single base site of a gene to be tested is homozygous or heterozygous can be determined by corresponding extended primers. Such as the following primers: Forward primer: 5 XXXXXXXXXX 3 '
  • X represents a base paired with a template (ie, a common genotype)
  • Y represents a base paired with a template (ie, a polymorphic genotype)
  • X, Y are unmodified bases
  • Z is a modified base.
  • the extension product can be obtained or the product has a label signal, and 5'XXXXXXXXXXX 'can not obtain the extension product or the product does not have a label signal, it means that the tested template is in the base
  • the homozygous genotype of the base is X; if the primer extension reaction is performed at 5′ ⁇ : 3 ′, an extension product or a product with a label signal can be obtained, and 5 ⁇ ⁇ 3 'can not get an extension product or the product without a label signal , It means that the tested template is homozygous for this base site; if both primers have products, it means that the base site is a heterozygous polymorphic genotype.
  • the invention provides a new method for determining a gene sequence.
  • the method is characterized by directness, sensitivity, simplicity, and speed.
  • the use of the present invention for determining the sequence of a gene does not require that the template to be tested is pre-amplified. Therefore, the direct base sequencing technology created by the present invention can be widely applied to the detection of single base polymorphisms.
  • the method will enable rapid gene sequence determination of biological samples that require high reliability and high sensitivity.
  • primer design from the mouse genome take any length of DNA of 217 bases, sequences bad '] is: cccaagatat ctgagaattc tcagcagcct tccatttaga agggtgttgt tgtctctgag gcaaaaccac atttcttacc gcacaactag agactgagac cagtttctct cattgtcatt gctgctcaga gccagcagaa aagcactcat gacacacact tagaataata gtgcatctga gccaggactg cccttggggt ccattcagct gttc gene region
  • a primer set is designed upstream and downstream of the segment: two forward primers (ie, a primer sub-collection) and a reverse primer. The length of the product of the reverse primer and the forward primer is 217 base pairs, which is completely the same as
  • the perfectly matched forward primer sequence is:
  • the incompletely matched forward primer sequence is:
  • the reverse primer sequence ⁇ ' is: 5'gaaacagctgaatggacccaa3'
  • Primer extension reaction The amplified forward primer used in the experiment is a base-specific primer.
  • the above primer and the reverse primer are respectively added to a primer extension reaction system containing a Deep Vent + enzyme and a Deep Vent- enzyme to perform a primer extension reaction.
  • the conditions of the extension reaction are: first melting at 95 degrees for 5 minutes, then melting at 95 degrees for 10 seconds, 56 degrees for 30 seconds for annealing, 72 degrees for 1 second for extension, and 30 cycles.
  • primers that are not perfectly matched to the template use 3 'to 5'
  • a polymerase with a dicerase activity is subjected to a primer extension reaction
  • its product can be digested by EcoR I
  • a polymerase that does not have a 3 'to 5' exonuclease activity is used for a primer extension reaction
  • the product cannot be digested by EcoR I.
  • the sequence of the product of the Deep Vent-enzyme-directed extension reaction is 5'AGT CCT CTC CTA TCC CAA GAT ATC TGA CAA TTC TTGGGTCCATTCAGCTGTTTC 3 '.
  • primer extension products produced by polymerases without 3 'to 5' exonuclease activity cannot alter primer sequences
  • primer extension products produced by polymerases with 3 'to 5' exonuclease activity can The sequence is changed to make it complementary to the template sequence; if a polymerase with 3 'to 5' exonuclease activity can reasonably be used to distinguish the pairing situation during primer extension reaction, the template can be read accurately from the primer sequence the sequence of.
  • Primer design taken from mouse genomic DNA length of 217 bases, sequences bad '1 ⁇ cccaagatat ctgagaattc tcagcagcct tccatttaga agggtgttgt tgtctctgag gcaaaaccac atttcttacc gcacaactag agactgagac cagtttctct cattgtcatt gctgctcaga gccagcagaa aagcactcat gacacacact tagaataata gtgcatctga gccaggactg cccttggggt ccattcagct gttc of SNP analysis
  • two forward primers and a reverse primer are designed upstream and downstream, respectively.
  • the product length of the reverse primer and the forward primer is 217 ⁇ base pairs.
  • Forward primers include three-terminal unlabeled primers 5'atcccaagatatctgagaatt3 'and three terminal 3 [E ⁇
  • Primer extension reaction The above-mentioned forward primer and reverse primer are respectively added to a primer extension reaction system containing a Deep Vent + enzyme and a Deep Vent- enzyme to perform a primer extension reaction, and the conditions of the primer extension reaction are: first melting 95 ° C for 5 minutes, then melted at 94 ° C for 40 seconds, annealed at 58.9 ° C for 30 seconds, 72 ° extended for 30 seconds, and cycled 25 times.
  • the primer extension reaction at the 3 'end of the radiolabeling has fewer extension products of primers that are incompletely paired than those of primers that are completely paired. Quantitative radiometric measurement showed that the extension product of the incompletely paired primer had lost the radiolabeled signal, which was 91 cpm, which was no difference from the blank control; while the extension product of the perfectly paired primer retained the radiolabel, which was 1019 cpm, ten times higher than the blank the above.
  • the primer extension product contains a detectable label signal; when the template is not paired with the primer, the label signal is lost as the three terminal bases are cleaved by the three exonuclease, so the primer Although it can be extended, the extension product has no obvious labeling signal.
  • the combination of Deep Vent + enzyme and Rox fluorescent three-terminal labeled primers also has a good recognition effect on single bases.
  • Primer design taken from a human genomic DNA length of 217 bases, sequences bad 1 J is: cccaagatat ctgagaattc tcagcagcct tccatttaga agggtgttgt tgtctctgag gcaaaaccac atttcttacc gcacaactag agactgagac cagtttctct cattgtcatt gctgctcaga gccagcagaa aagcactcat gacacacact tagaataata SNP analysis gtgcatctga gccaggactg cccttggggt ccattcagct gttc with a particular For the gene segment, seven forward primers and one reverse primer (ie, a set of primers) are designed upstream and downstream, respectively.
  • the forward primer is a base-specific primer, which contains a paired primer and a single base
  • the perfectly matched primer sequence is 5'atcccaagatatctgagaattc3 ';
  • sequences of primers that are incompletely matched from -1 to -6 base positions indicated by capital letters are:
  • the reverse primer has no combing modification, and its sequence is 5'gaaacagctgaatggacccaa3 '. After the primers are designed, they will be synthesized by MWG USA.
  • Primer extension reaction The above primers and reverse primers are respectively added to a primer extension reaction system containing a Deep Vent + enzyme and a Deep Vent- enzyme to perform a primer extension reaction.
  • the conditions of the primer extension reaction are: the first melting point is 95 degrees 5 minutes, then Melt 94 degrees and 40 seconds, anneal for 30 seconds, 72 degrees and extend for 30 seconds, and cycle 25 times.
  • the 3 'sulfur-modified 3' end or sub 3 'end single base unpaired primers are polymerized immature due to resistance to exonuclease digestion Termination, that is, the effect of closing the DNA polymerization reaction, cannot be extended without obtaining an extension product. It proves that the present invention can be used for gene sequence analysis.
  • primer design from mouse genomic DNA to take a length of 217 bases, sequences bad 1] is: cccaagatat ctgagaattc tcagcagcct tccatttaga agggtgttgt tgtctctgag gcaaaaccac atttcttacc gcacaactag agactgagac cagtttctct cattgtcatt gctgctcaga gccagcagaa aagcactcat gacacacact tagaataata SNP analysis gtgcatctga gccaggactg cccttggggt ccattcagct gttc with a particular Gene segment, divided upstream and downstream Do not design two forward primers and one reverse primer.
  • the forward primer is a base-specific primer, which contains a paired primer and a single base incompletely matched primer:
  • the sequence of the perfectly matched primer is 5atcccaagatatctgagaattc3, and the single base at the 3 'end is not matched.
  • the sequence of the primer is 5atcccaagatatct gagaattG3; the three ends of the perfectly matched primer and the unpaired base are all sulfurized modified; the reverse primer has no sulfurized modification, and its sequence is 5gaaacagctgaatggacccaa3 ', the length of the primer extension product is 217 base pairs, and the primer design After that, it will be synthesized by MWG USA.
  • Primer extension reaction The above primers and reverse primers are respectively added to a primer extension reaction system containing a Deep Vent + enzyme and a Deep Vent- enzyme to perform a primer extension reaction.
  • the conditions of the primer extension reaction are: the first melting point is 95 degrees 5 minutes, then melted 94 degrees and 40 seconds, annealed for 30 seconds, the temperature was in the range of 46-66 degrees, 72 degrees was extended for 30 seconds, and the cycle was 25 times.
  • Deep Vent + enzymes with 3 'to 5' exonuclease ability extend only 100% accurate primers, and incompletely matched primers do not Extended, while Deep Vent-enzymes without 3 'to 5' exonuclease ability showed resolution of the primer's 3 'end sequence only at greater than 62 degrees, indicating that the method can be used at a wide range of annealing temperatures in a similar manner. Or under the same reaction conditions to analyze the gene sequence.
  • Primer design The forward primer sequence corresponding to wild type allele C in the human genomic DNA neurodeafness-related SNP site gene segment is 5'caa cat cgt gga ctg cta cat tgc cc3 ', which 5'gtg aag reverse primer sequence Att ttc ttc ttg gta ggt cg3 '.
  • the forward primer sequence corresponding to the deafness mutation site T is 5'caa cat cgt gga ctg cta cat tgc ct3', and its reverse primer sequence is 5'gtg aag att tc ttc ttg gta ggt ca3 '.
  • the 3' end of the forward primer must be thinned.
  • the primers are designed and delivered to Shanghai Bioengineering Company for synthesis.
  • Primer extension reaction The above two sets of forward primers and reverse primers are respectively added to a primer extension reaction system containing pfu enzyme and Taq enzyme without 3 'to 5' exonuclease activity to perform a primer extension reaction, and a primer extension reaction The conditions are: first melting at 95 ° C for 5 minutes, then melting at 94 ° C for 40 seconds, annealing at 56 ° C for 40 seconds, extending at 72 ° C for 30 seconds, and cycling 30 times.
  • PCR products were electrophoretic separated using a 2.5% agarose gel pre-containing ethidium bromide under a DC electric field of 10 V / cm.
  • the electrophoresis solution was 0.5x TBE buffer.
  • FIG. 8 when a Taq enzyme lacking 3 ′ to 5 ′ exonuclease activity is used, although the template used is homozygous for a wild-type site, wild-type allele site-specific primers and point mutation alleles All site-specific primers can be extended.

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Abstract

La présente invention se rapporte à un procédé permettant d'effectuer une analyse de séquence de gènes au moyen d'une polymérase dotée d'une activité de 3'-5' exonucléase, qui comprend les étapes suivantes: a. préparation d'amorces spécifiques ayant leur extrémité terminale 3' modifiée; b. traitement de la polymérase dotée d'une activité de 3'-5' exonucléase pour former une séquence activatrice « marche/arrêt » avec les amorces modifiées, et mise en oeuvre d'une réaction sélective d'extension des amorces avec participation de ladite séquence activatrice « marche/arrêt »; c. détection de l'image des produits d'extension des amorces et analyse des séquences. Avec la séquence activatrice du procédé de la présente invention, la précision est meilleure qu'avec les réactions d'extension d'amorces mises en oeuvre de manière classique dont la médiation est assurée par une polymérase non dotée d'une fonction de correction d'épreuves. Cette séquence activatrice peut également être utilisée pour une analyse directe de séquences inconnues, et elle ne nécessite pas l'existence d'un segment de séquence connue lors du séquençage effectué conformément au procédé de la présente invention. Chaque signal obtenu conformément à ce procédé fournit des informations pour une ou plusieurs séquences de base. Le procédé de la présente invention constitue une approche d'analyse à haut rendement pour l'analyse des polymorphismes mononucléotidiques et l'analyse de séquences de gènes.
PCT/CN2003/000695 2002-08-19 2003-08-19 Utilisation d'une polymerase dotee d'une activite de 3'-5' exonuclease pour effectuer une analyse de sequence de genes WO2004016806A1 (fr)

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JP2004528265A JP2005535336A (ja) 2002-08-19 2003-08-19 ３’−５’エキソヌクレアーゼを有するポリメラーゼを用いる配列決定方法
EP03787581A EP1536019A4 (fr) 2002-08-19 2003-08-19 Utilisation d'une polymerase dotee d'une activite de 3' a 5' exonuclease pour effectuer une analyse de sequence de genes
AU2003257798A AU2003257798A1 (en) 2002-08-19 2003-08-19 Using a polymerase with 3' to 5' exonuclease activity to perform gene sequence analysis

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CN02129166.7 2002-08-19
CN02129166A CN1414110A (zh) 2002-08-19 2002-08-19 利用具有3'至5'外切酶活性的多聚酶进行基因序列分析

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CN104561248A (zh) * 2013-10-22 2015-04-29 常州金麦格生物技术有限公司 用于检测靶核酸的引物和其应用
CN106055924B (zh) * 2016-05-19 2019-02-01 完美（中国）有限公司 微生物操作分类单元确定和序列辅助分离的方法和系统
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EP1536019A1 (fr) 2005-06-01
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US20060172307A1 (en) 2006-08-03
AU2003257798A1 (en) 2004-03-03

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